Purification and properties of isopenicillin N epimerase from Streptomyces clavuligerus

Isopenicillin N epimerase, which catalyzes conversion of isopenicillin N to penicillin N, has been purified to electrophoretic homogeneity from the cell-free extract of Streptomyces clavuligerus by a procedure involving ammonium sulfate fractionation and chromatographies with DE-52, DEAE Affi-gel blue, Sephadex G-200, calcium phosphate-cellulose, and Mono Q. The purified epimerase is monomeric with a molecular weight of 47,000 or 50,000 as estimated by SDS-polyacrylamide gel electrophoresis or gel filtration, respectively. The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of protein, and shows absorption maxima at 280 and 420 nm. The epimerase catalyzes the complete 'racemization' on both the L-alpha-aminoadipyl side-chain of isopenicillin N and the D-alpha-aminoadipyl side-chain of penicillin N, so that an approximately equimolar mixture of the two penicillins is produced. The mixture is not truly racemic, since these penicillins are diastereomers rather than optical isomers. The chemical modification of primary amino groups of the epimerase by fluorescamine results in a great loss of the enzyme activity. The activity of purified enzyme is partially stimulated by the addition of sulfhydryl compounds. The activity is strongly inhibited by sulfhydryl group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.     

A branched-chain amino acid aminotransferase from the rumen ciliate genus Entodinium.

A branched-chain amino acid aminotransferase was extracted from rumen ciliates of the genus Entodinium and was partially purified by Sephadex G-200, DEAE-cellulose and DEAE-Sephasex A-50 column chromatography. The purified enzyme was active only with leucine, isoleucine and valine, and required pyridoxal phosphate as cofactor. The amino acids competed with each other as substrates. The enzyme had optimal activity at pH 6.0 in phosphate buffer. The Km values for the substrates and cofactor are as follows: 1.66 for leucine; 0.90 for isoleucine; 0.79 for valine; 0.29 mM for alpha-ketoglutarate; and 0.1 muM for pyridoxal phosphate. Enzyme activity was inhibited by rho-chloromercuribenzoate and HgCl2. Gel filtration indicated the enzyme to have a molecular weight of 34,000.     

Studies on a 2,3-diaminopropionate: ammonia-lyase from a Pseudomonad.

2,3-Diaminopropionate:ammonia-lyase, an induced enzyme in a Pseudomonas isolate, has been purified 40-fold and found to be homogeneous by disc gel electrophoresis and by ultracentrifugation. Some of its properties have been studied. The optimum pH and temperature for activity are 8 and 40 degrees C, respectively. The enzyme shows a high degree of substrate specificity, acting only on 2,3-diaminopropionate; the D-isomer is only one-eighth as effective as the L-form. L-Homoserine and DL-cystathionine are not substrates, and 3-cyanolalanine does not inhibit its activity. It is a pyridoxal phosphate enzyme which requires free enzyme sulphhydryls for activity. The Km values for L-2,3-diaminopropionate and pyridoxal phosphate are 1mM and 25 muM, respectively. The molecular weight of the enzyme is about 80 000 as determined by gel filtration. On treatment with 0.5M urea or guanidine by hydrochloride, the enzyme dissociates into inactive subunits with an approximate molecular weight of 45 000. One mole of the active enzyme binds one mole of pyridoxal phosphate. The bacterial enzyme seems to be quite different in many of its properties from the rat liver enzyme which also exhibits the substrate specificity of cystathionine gamma-lyase.     

Purification and Properties of 1-AminocycIopropane-l-carboxylate Synthase of Mesocarp of Cucurbita maxima Duch. Fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, which formsAGC from S-adenosylmethionine (SAM), was purified to homogeneityfrom sliced and aged mesocarp tissue of Cucurbita maxima Duch.cv Ebisu fruits, and its enzymatic properties were determined.The specific activity of the purified enzyme was 220 mU/mg proteinat 30°C at 50 µM SAM. Native ACC synthase has a relativemolecular mass of 160 ± 10 kDa and consisted of two subunitsof about 84±3 kDa. S-adenosylhomocysteine (SAH), S-methylmethionine(SMM) and L-methionine did not serve as substrate. The enzymereaction was competitively inhibited by aminoethoxyvinylglycine(AVG) (Ki, 2.5 µM), aminooxyacetic acid (Ki, 40 µM)and SAH (Ki, 30 µM). The reaction was also strongly inhibitedby semicarbazide, and less effectively by homocysteine. Theenzyme was rapidly inactivated by its substrate, SAM in thepresence of pyridoxalphosphate (PLP), but in the absence ofPLP, SAM-induced inactivation was much slower. Inactivationdid not occur by SAH and SMM, SAM analogs without substrateactivity. Pyridoxal phosphate was an essential cofactor to beadded to a reaction mixture for maximum activity, but an enzymepreparation from which pyridoxal phosphate was removed by SephadexG-25 gel filtration exhibited one-eighth activity which wasinhibited by semicarbazide, this indicating that a small amountof pyridoxal phosphate is firmly bound to the enzyme. (Received May 6, 1986; Accepted May 20, 1986)     

Partial purification and properties of frog liver tyrosine aminotransferase.

Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5'-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics.     

GABA-TRANSAMINASES OF HUMAN BRAIN AND PERIPHERAL TISSUES—KINETIC AND MOLECULAR PROPERTIES

Abstract— Kinetic experiments with 4-aminobutyrate-2-ketoglutarate transaminase (GABA-T), partially purified from human brain tissue, supported a Bi Bi Ping-Pong type of enzyme mechanism in which the enzyme oscillates between forms bound to pyridoxal phosphate and pyridoxamine phosphate. Extrapolated K _m values were 0.31 m m for γ-aminobutyrate, 0.16 m m for α-ketoglutarate, and 3.8 μ m for pyridoxal phosphate. Very similar kinetic parameters were observed with rat brain enzyme. Apparent molecular weight of human GABA-T by gel filtration was 70,000 ± 3000. Electrofucusing experiments indicated a single ionic form with isoelectric pH = 5.7. Enzyme activity was inhibited by Tris, halides, cadmium and cupric ions, and known GABA-T inhibitors.
GABA-transaminating enzymes isolated from human kidney and liver were found to be similar to the brain enzyme with respect to substrate affinities, cofactor requirements, isoelectric pH values, molecular weights, and response to inhibitors.     

Degradative acetolactate synthase of Bacillus subtilis: purification and properties.

A degradative acetolactate synthase (acetolactate pyruvate-lyase [carboxylating], EC 4.1.3.18) from Bacillus subtilis has been partially purified and characterized. The synthesis of the enzyme was induced by growth of cells in minimal medium plus isobutyrate or acetate. The enzyme was partially purified by ammonium sulfate fractionation, gel filtration, and hydroxyapatite chromatography. The pH optimum of the purified enzyme was 7.0 in phosphate buffer. When assayed in phosphate buffer (pH 7.0), activity was stimulated by acetate and inhibited by sulfate. When assayed in acetate buffer (pH 5.8), activity was inhibited both by sulfate and phosphate. Michaelis-Menten kinetics was observed when the enzyme was assayed in phosphate buffer (pH 6.0 or 7.0), and inhibition by sulfate was competitive and activation by acetate was noncompetitive. When assayed in acetate buffer (pH 5.8), nonlinear Lineweaver-Burk plots were obtained; inhibition by phosphate appeared to be competitive and that by sulfate was of the mixed type. The approximate molecular weight of the purified enzyme was 250,000 as determined by gel filtration.     

Cysteine synthase from rape leaves.

Cysteine synthase [O-Acetyl-L-serine acetate-lyase (adding hydrogen-sulfide) EC 4.2.99.8] has been highly purified from the extract of rape, Brassica chinensis var. Komatsuna. The purified preparation appeared to be homogeneous on Sephadex G-100 gel filtration and dodecylsulfate-polyacrylamide gel electrophoresis, showing a molecular weight of about 62,000. The latter method also suggested that this enzyme was composed of two identical subunits. The enzyme contained 2 moles of pyridoxal phosphate per mole of enzyme.     

NAD+-specific glycerol 3-phosphate dehydrogenase from developing castor bean endosperm

l-Glycerol 3-phosphate dehydrogenase has been isolated and partially purified from the endosperm of developing castor beans. The enzyme is entirely cytosolic and is not found in the plastid fraction. No activity was found in germinating castor beans. The pH optimum for the reduction of dihydroxyacetone phosphate is 8.1 and is 9.6 for the reverse reaction. The molecular weight determined by gel filtration chromatography is between 71,000 and 83,000. Both substrates show substrate inhibition at concentrations about 13 μm for NADH and 400 μm for dihydroxyacetone phosphate. Substrate interaction kinetics gave limiting K_m values of 2.7 and 35.5 μm for NADH and dihydroxyacetone phosphate, respectively. Substrate interaction and product inhibition kinetics were consistent with an ordered sequential mechanism with NADH being the first substrate to bind and NAD⁺ being the last product to dissociate.     

l-Ornithine:2-Oxoacid Aminotransferase from Squash (Cucurbita pepo, L.) Cotyledons: Purification and Properties

ORNITHINE: 2-oxoacid aminotransferase (EC 2.6.1.13) has been purified over 400-fold with a total recovery of 14% from acetone powders of cotyledons of germinating squash (Cucurbita pepo, L.) seedlings. The pH optimum of the transamination between l-ornithine and alpha-ketoglutarate is 8 and the Michaelis constants are 4.7 mm and 6.3 mm, respectively. The enzyme has a molecular weight of 48,000 as determined by gel filtration. The reaction is essentially specific for alpha-ketoglutarate as the amino group acceptor. The enzyme is inhibited very strongly by hydroxylamine, and less severely by NaCN and isonicotinylhydrazide. No inhibition is observed in the presence of 10 mml-cysteine. The energy of activation is 7.6 kcal/mole. The stability of the enzyme preparation is enhanced by the presence of dithioerythritol and glycerol. The enzyme activity of the most purified fraction is stimulated 30% by the addition of pyridoxal phosphate; however, the evidence for the unequivocal involvement of pyridoxal phosphate was inconclusive.     

STUDY ON THE INHIBITION OF BRAIN GLUTAMATE DECARBOXYLASE BY PYRIDOXAL PHOSPHATE OXIME-O-ACETIC ACID

The kinetics of the inhibition of mouse brain glutamate decarboxylase by pyri-doxaI-5′-phosphate oxime-O-acetic acid (PLPOAA) was studied. The inhibition was noncompetitive with regard to glutamic acid; it could be partially reversed by pyridoxal phosphate, but only when the concentration of the latter in the incubation medium was higher than that of pyridoxal-5′-phosphate oxime-O-acetic acid. The inhibition produced by aminooxyacetic acid, which is remarkably greater than that produced by PLPOAA, was also partially reversed only when an excess of pyridoxal phosphate was added. Both in the presence and in the absence of a saturating concentration of pyridoxal phosphate, the activity of the enzyme was decreased by PLPOAA at a 10^?4m concentration to a value of about 50 per cent of the control value obtained without added coenzyme. This activity could not be further reduced even when PLPOAA concentration was increased to 5 × 10^?3m . This same minimal activity of glutamate decarboxylase was obtained after dialysis of the enzymic preparation, or after incubation with glutamic acid in the cold followed by filtration through Sephadex G-25. The addition of pyridoxal phosphate to the dialysed or glutamic acid-treated enzyme restored the activity to almost the control values. PLPOAA did not affect the activity of glutamate decarboxylase from E. coli or that of DOPA decarboxylase and GABA transaminase from mouse brain. To account for the results obtained it is postulated that brain glutamate decarboxylase has two types of active site, one with firmly bound, non-dialysable pyridoxal phosphate and the other with loosely bound, dialysable coenzyme; PLPOAA behaves as a weak inhibitor probably because it can combine mainly with the loosely bound coenzyme site, while aminooxyacetic acid is a potent inhibitor probably because it can block both the ‘loosely bound coenzyme’ and the ‘firmly bound coenzyme’ sites.     

A Branched-chain Amino Acid Aminotransferase from the Rumen Ciliate Genus Entodinium

A branched-chain amino acid aminotransferase was extracted from rumen ciliates of the genus Entodinium and was partially purified by Sephadex G-200, DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. The purified enzyme was active only with leucine, isoleucine and valine, and required pyridoxal phosphate as cofactor. The amino acids competed with each other as substrates. The enzyme had optimal activity at pH 6.0 in phosphate buffer. The K_m values for the substrates and cofactor are as follows: 1.66 for leucine; 0.90 for isoleucine; 0.79 for valine; 0.29 mM for α-ketoglutarate: and 0.1 μM for pyridoxal phosphate. Enzyme activity was inhibited by p-chloromercuribenzoate and HgCl₂. Gel filtration indicated the enzyme to have a molecular weight of 34,000.     

Histidine decarboxylase. Purification from fetal rat liver, immunologic properties, and histochemical localization in brain and stomach

Histidine decarboxylase from fetal rat liver was purified to near-homogeneity. The purified enzyme has a molecular weight of 210,000, and appears to contain two subunits with molecular weights of 145,000 and 66,000, respectively. The enzyme is inhibited by heavy metals such as Hg2+ and Zn2+ and sulfhydryl-reactive compounds such as 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme is partially dependent on exogenous pyridoxal phosphate. Extensive dialysis results in 50% loss of enzyme activity which can be fully recovered by adding pyridoxal phosphate. Affinity of pyridoxal phosphate for the apoenzyme is 0.1 microM at pH 6.8. Antibody against purified histidine decarboxylase was raised in rabbits. The antibody has been employed in immunohistochemical studies to visualize histidine decarboxylase containing cells and neuronal processes in rat stomach and brain, respectively. Immunologic studies indicate that histidine decarboxylase from brain, gastric mucosa, and fetal rat liver share common antigenic properties.     

Purification and characterization of glutamate decarboxylase from Solanum tuberosum

Glutamate decarboxylase has been purified from potato tubers. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Gel filtration on Sephadex G-200 gave a relative molecular mass Mr, of 91 000 for the native enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a subunit Mr of 43 000. Thus the enzyme appears to be a dimer of identical subunits. It has 2 mol pyridoxal 5'-phosphate/mol protein, which could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25. The enzyme has an absorption maximum at 370 nm in sodium phosphate buffer, pH 5.8. Reduction of the enzyme with sodium borohydride abolished the absorption maximum at 370 nm with attendant loss of catalytic activity. The enzyme exhibited pH-dependent spectral changes. The enzyme was specific for L-glutamate and could not decarboxylate other amino acids tested. The enzyme was maximally active at pH 5.8 and a temperature of 37 degrees C. Isoelectric focussing gave a pI of 4.7 Km values for L-glutamate and pyridoxal 5'-phosphate were 5.6 mM and 2 microM respectively. Thiol-directed reagents and heavy metal ions inhibited the enzyme, indicating that an -SH group is required for activity. The nature of the functional groups at the active site of the enzyme was inferred from competitive inhibition studies. L-Glutamate promoted inactivation of the enzyme caused by decarboxylation-dependent transamination was demonstrated. The characteristics of potato enzyme were compared with enzyme from other sources.     

Biosynthesis of branched-chain amino acids in Schizosaccharomyces pombe: regulatory properties of threonine deaminase

Biosynthetic threonine deaminase (TD) from Schizosaccharomyces pombe has been partially purified from crude extracts by treatment with protamine sulfate, ammonium sulfate precipitation, and gel filtration through Sephadex G-25. In both crude extracts and purified preparations, TD showed marked stimulation by pyridoxal phosphate. A pH optimum for activity was found at pH 9.0, whereas the inhibition caused by the natural feedback inhibitor, l-isoleucine, was maximal at pH 7.4. l-Threonine exhibits homotropic cooperative effects at low pH (7.0-8.0), which are eliminated at pH 9.0, and the affinity for substrate (in terms of K(m)) increased with increasing pH. Enzyme activity could be completely inhibited by isoleucine over a pH range of 7.4 to 9.0; the amount of isoleucine required for 50% inhibition increased with increasing pH. Isoleucine inhibition was pseudocompetitive with respect to substrate and increased the cooperative effects of threonine. l-Valine was found to reverse isoleucine inhibition; it also activated the enzyme in a pH range of 7.0 to 8.0 by eliminating the cooperative effects of threonine, thus normalizing the substrate saturation curves at these pH values. l-Leucine was shown to be a competitive inhibitor with respect to threonine, and to be able partially to reverse isoleucine inhibition. Treatment of TD with mercurials did not result in desensitization to isoleucine inhibition. However, at pH 10, virtually no sensitivity of the enzyme to isoleucine was observed while activity remained strong, which suggests the existence of separate sites on the TD molecule for binding threonine and isoleucine. A tentative model is presented which unifies the kinetic results reported here in terms of the interactions of TD with its effector molecules.     

Characterization of intermediate-molecular-weight acid phosphatase from bovine kidney cortex

The distribution of acid phosphatases of intermediate molecular weight was determined in various mammalian tissues. The intermediate-molecular-weight acid phosphatases (designated P-II-1 and 2) comprised about 25% of the p-nitrophenyl phosphatase activity in the supernatant of bovine kidney cortex homogenate. The P-II-1 and 2 purified 2,000 fold showed the pI values of 5.9 and 5.7, respectively, on isoelectric focusing. Apparent molecular weights of both P-II-1 and 2 were estimated to be 42,000 by Sephadex G-100 gel filtration and 44,000 by SDS-polyacrylamide disc gel electrophoresis. Both the enzymes catalyzed the hydrolysis of a wide variety of natural phosphomonoesters, except for the phosphoproteins phosphoserine and o-phosphocholine. The enzymes showed a high activity on pyridoxal phosphate, beta-glycerophosphate, and 2'-AMP. The optimum activity pH was near 5 with p-nitrophenyl phosphate, but was shifted to the neutral range when pyridoxal phosphate was the substrate. The cations Hg2+ and Ag+ had a marked inhibitory effect. Neither enzyme was inhibited significantly by L-(+)-tartrate or pCMB. The two other types of acid phosphatases, the high-molecular-weight (designated P-I) and low-molecular-weight (designated P-III), were also purified to homogeneity from bovine kidney cortex, and were compared with P-II from several aspects including substrate specificity and susceptibility to various compounds.     

Lysyl oxidase: evidence that pyridoxal phosphate is a cofactor

Both crude and partially purified preparations of embryonic chick aortic lysyl oxidase tend to gradually lose enzymic activity when illuminated, or when urea is removed by dialysis. Full activity is restored to such preparations by dialysis versus low concentrations of pyridoxal 5′-phosphate prior to assay. Upon treatment with potassium cyanide or semicarbazide, purified embryonic chick aortic lysyl oxidase gives rise to fluorescent derivatives. The fluorescence spectrum of the semicarbazide adduct closely resembles that of pyridoxal phosphate semicarbazone. A preliminary ultraviolet/visible spectrum of bovine aortic lysyl oxidase is also presented; this shows features which add to the existing evidence that lysyl oxidase contains an essential pyridoxal phosphate cofactor.     

Ornithine decarboxylase from calf liver. Purification and properties

Ornithine decarboxylase (ODC) was purified 25000-fold from calf liver to apparent homogeneity by methods developed to circumvent the lability of the enzyme. Appropriate ratios of sample protein applied to column size and/or gradient size were derived for each purification procedure (ion-exchange, gel filtration ahd hydroxylapatite chromatography, electrophoresis, and thiol affinity chromatography) to maintain enzymatic activity. The enzyme was labile to dilution at all steps of the purification; the inclusion of poly(ethylene glycol) or additional protein decreased but did not eliminate the activity loss. The purified enzyme had a Stokes radius of 3.14 and a molecular weight of 54000. The Km for ornithine was 0.12 mM, and pyridoxal phosphate was 2.0 microM; the pH optimum for the decarboxylation reaction was 7.0. Analysis by sievorptive ion-exchange chromatography indicated the presence of three ionic forms. In the presence of Tris-barbital buffer containing thioglycolic acid, the ODC preparation assumed an apparent molecular weight of 100000 and a Stokes radius of 4.5 and retained full catalytic activity.     

Brain pyridoxal kinase. Purification and characterization

Pyridoxal kinase has been purified 9000-fold from sheep brain. The purification procedure involves ammonium sulphate fractionation, DEAE-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration. The final chromatography step yields a homogeneous preparation of high specific activity with a pI of 5. The molecular mass of the native enzyme was estimated to be approximately 80 kDa by 10-25% gradient polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration. The subunit molecular mass was determined by sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis to be 40 kDa compared with a series of molecular mass standards. This indicates that pyridoxal kinase is a dimeric enzyme. Further results obtained from electron microscopy, using a negative staining technique, provide evidence that pyridoxal kinase exists as a dispherical subunit structure.